Borrelia burgdorferi is a highly motile and invasive spirochete pathogen causing Lyme disease, the most common vector-borne infection in the United States. Periplasmic flagella, the main organelles for motility, are essential for the distint morphology, motility and infectious life cycle of B. burgdorferi. The periplasmic flagella are distinct from the external flagella in the model systems Escherichia coli and Salmonella enterica, as they are enclosed within the outer membrane and their flagellar motors are considerably larger and more complex. Importantly, owing to its small cell diameter and highly ordered flagellar motors at the cell tip, B. burgdorferi is emerging as a unique paradigm for in situ structural analysis of the periplasmic flagella by employing the ground-breaking methodology of cryo-electron tomography (cryo-ET). During the previous funding period, we have generated a large B. burgdorferi library of over 40 different flagellar and chemotaxis mutants in collaboration with Drs. Steven Norris, Md Motaleb, Chunhao Li and Nyles Charon. Significant progress has been made in understanding the unique periplasmic flagella and their dramatic impacts in the unique spirochetal motility and morphology. The objective of this application is to understand three fundamental aspects of the periplasmic flagella: 1) the structural basis of the flagellar rotation; 2) the flagellar switching mechanism; and 3) the structure and mechanism of the flagellar type III secretion apparatus. Together with genetic and biochemical approaches, cryo-ET will be utilized to determine the structure/function relationship of the spirochetal flagellar motor in native cellular environment.